Method of quantifying peptide-derivative libraries using phage display

ABSTRACT

The present application provides a method of quantifying an amount of a derivatized peptide displayed on a phage by phage display, the method comprising: providing a phage containing a target peptide thereon; reacting the phage containing the target peptide with a first reagent to derivatize the target peptide to form a derivatized peptide, reacting the derivatized peptide with a capture agent comprising a detection marker, thereby incorporating the detection marker within the derivatized peptide; and determining an amount of the detection marker, thereby quantifying the amount of the derivatized peptide dis-played on the phage. A kit comprising a capture agent compris-ing a detection marker for quantifying the phage displayed derivatized peptides is also provided.

FIELD

The present application pertains to the field of recombinant proteintechnology. More particularly, the present application relates to amethod of quantifying derivatized peptide libraries using phage display.

BACKGROUND

The generation of libraries of small molecules and selection of thosemolecules that bind uniquely to a target of interest is important fordrug discovery. The production of genetically-encoded libraries, inwhich each library member is linked to an information template, such asDNA or RNA, makes it possible to process large chemical librarieswithout separating individual library members into individual solutionsand reaction vessels. One can select target molecules from mixtures ofgenetically-encoded molecules and identify or amplify the selectedmolecule of interest using its information template.

Phage display is one example of a genetically-encoded library. Phagedisplay is a well known technique used in the analysis, display andproduction of protein antigens, especially human proteins of interest.Phage display is a process during which the phage, a bacterial virus, ismade to expose or “display” different peptides or proteins includinghuman antibodies on its surface. Through genetic engineering, peptidesor proteins of interest are attached individually to a phage cellsurface protein molecule (usually Gene III protein, g3p). In such aphage population (phage library), each phage carries a gene for adifferent peptide or protein-g3p fusion and exposes it on its surface.Through a variety of selection procedures, phages that “display” bindersto specific target molecules of interest can be identified and isolated.These binders can include interaction partners of a protein to determinenew functions or mechanisms of function of that protein, peptides thatrecognize and bind to antigens (for use in diagnosis and therapeutictargeting, for example), and proteins involved in protein-DNAinteractions (for example, novel transcription factors).

The phage display technique can be very useful in discovery anddevelopment of pharmaceutical and/or diagnostic products. In phagedisplay the entire phage binds and can be eluted from an immobilizedtarget molecule. Since the phage remains infective it can inject its DNAinto bacterial cells and is amplified. The main limitation of phagedisplay, however, is the occurrence of non-specific adsorption of phagesduring the binding stage, which necessitates enrichment over severalrounds and individually tailored washing and elution conditions. Phagedisplay methods are usually restricted to the production of libraries,which can be encoded by direct DNA-RNA-protein information transfer.These methods are typically limited to linear sequences of peptides,made of only 20 natural amino acids.

Typically, the amplification of libraries of peptides on the surface ofthe phage requires an in vitro translation system, in which DNA ismodified to express the displayed peptides of interest. The generationand use of such translation systems can be expensive and time consuming.The use of self-replicating species such as phage or bacteria simplifiesamplification of libraries because each library member is amplified“spontaneously”, when given the appropriate resources. For example, forphage displayed libraries, adding one phage to a simple culture brothwith bacteria can produce an arbitrarily large population of phage for avery low cost.

Several methods exist which involve conversion of libraries ofphage-displayed polypeptides to libraries of peptide derivatives.Typically, these methods use organic synthesis on the peptides to makepeptide derivatives. The characterization and improvement of reactionyields is an important cornerstone of organic synthesis. Bulkbiochemical methods, such as western blot and mass spectrometry, areoften used, to quantify the amount of product obtained or to determinethe success of generating the desired reaction products. However, in theabsence of this characterization, the synthesis cannot be claimed to bereliable or reproducible. Reactions used for synthesis of such librariesof peptide derivatives have typically been validated using one phageclone or one purified peptide. The actual synthesis of libraries istypically done “blindly”, and the efficiency of such synthesis isunknown. The quality of the libraries generated by this method is, thus,usually unknown. While selection from these libraries can provide usefulnon-peptidic molecules, overall the efficiency of such selection isunclear.

Jesper at al. in U.S. Pat. No. 6,017,732, describe chemical modificationof point residues in antibodies displayed on phagemid. This patentdiscusses a limited set of reactions including the alkylation of a Cysresidue within an antibody, and subsequent detection with fluorescenceand radioactive probes. The technique described in Jesper is designedfor estimating the yield in large (>10¹⁰ copies) clonal populations ofphage. However, there is no teaching or suggestion of the quantificationor optimization of a derivative library synthesis.

U.S. Pat. No. 7,141,366 to Noren et al, describes the production ofphage with a unique chemical residue, selenocysteine (Sec), incorporatedin a specific location in a phage-displayed peptide. Phage-displayedpeptide libraries that contain Sec were generated, thus creatingmodified peptide libraries. Bulk biochemical methods, such as westernblot, were used to qualitatively characterize the chemical modificationsthat occur on the Sec residue. However, this method cannot be extendedto Sec-free peptide libraries, and, further, there is no teaching orsuggestion of the quantification or optimization of a derivative librarysynthesis.

US Patent Publication 2009/0137424 to Schultz et al, describes theproduction of phage with non-natural amino acids incorporated at aspecific location within a phage-displayed peptide using orthogonal tRNAand aminoacyl tRNA synthase. Phage-displayed peptide libraries thatcontain azido phenyl alanine (AzPhe) are generated. Bulk biochemicalmethods, such as western blot and mass spectrometry, are used toqualitatively characterize the chemical modifications that occur onAzPhe residues. No methods for quantification of chemical yields insingle or multi-step reactions in the synthesis of the library aredescribed, nor strategies for improvement of chemical yields.

U.S. Pat. No. 6,642,014 to Pedersen et al, describes the use of captureagents to select enzymes with improved or new activities. This patentdiscusses the generation of chemicals by enzyme catalysis, not by directchemical transformation. Specially-engineered phagemid is used andrequired to display both enzyme and substrate on the surface of phage.

US Patent Publication 2010/0317547 to Winter and Heinis, describesmethods for the generation of a library of bicyclic peptides displayedon phage from a linear peptide, which contains random amino acidsflanked by three cysteine residues.

Typically, and as indicated in the above references, the phage displaymethods known in the art are not designed for efficiency and success ofthe reactions during synthesis. These previous systems are oftencharacterized on purified protein from phage well after the derivatizingreaction has taken place. The yields of reactions on phage, and thequality and purity of the library are, thus, generally unknown.

Thus, there remains a need for quantitative characterization ofpeptide-derivative phage display to determine the quality of the phagedisplay procedure and a corresponding optimization of the procedure toensure optimal yields of reaction products.

This background information is provided for the purpose of making knowninformation believed by the applicant to be of possible relevance to thepresent application. No admission is necessarily intended, nor should beconstrued, that any of the preceding information constitutes prior artagainst the present invention.

SUMMARY

An object of the present application is to provide a method ofquantifying peptide-derivative libraries using phage display.

In accordance with one aspect of the present invention, there isprovided a method of quantifying an amount of a derivatized peptidedisplayed on a phage by phage display, the method comprising: providinga phage containing a target peptide displayed thereon; reacting thephage containing the target peptide with a first reagent to derivatizethe peptide to form a derivatized peptide, reacting the derivatizedpeptide with a capture agent comprising a detection marker, therebyincorporating the detection marker within the derivatized peptide; anddetermining an amount of the detection marker, thereby quantifying theamount of the derivatized peptide displayed on the phage.

The present application provides a highly sensitive method for thequantitative characterization of reaction yields on individual phageclones or for a library of phage. Ideally, the method is suited for thedetection of as little as 1 to 100 copies of molecules that have beenderivatized by the reactions described herein.

The present method can be employed with known techniques, includingdetection assays known in the art. Thus, the present method can offer anadvantage over prior phage display techniques, which rely on bulkbiochemical methods. Further, it has been found that the method of thepresent invention offers increased sensitivity for detecting derivatizedpeptides not afforded with prior phage display techniques and assaysused to detect displayed peptides.

In certain embodiments, the present method employs the reaction ofterminal functional groups on peptides presented on the surface of phageto incorporate detection markers. The detection markers are coupled withchemical moieties to form capture agents. These capture agents reactwith desired functional groups modified from peptide residues ofinterest. The detection markers can include, but not limited to, biotin,fluorescein and mannose. Other suitable detection markers known in theart may be used.

The present method provides an improvement over prior methods of phagedisplay because it permits an assay of the yields of the desired phagelibrary or sub-libraries using specific reaction conditions optimizedfor the particular library or sub-libraries. This is achieved bymodifying the functional groups of library members to possess uniqueproperties which are then assayed. These properties can includefluorescence or enzyme-linked quantification. Additionally, thederivative libraries can include macrocycles (which incorporate peptideresidues and a light responsive linker) or complex carbohydrate moieties(which cannot typically be incorporated via ribosomal synthesis orenzymatic synthesis), or the method can use multiple reactions forquantitative synthesis of libraries of complex cyclic and poly-cyclicpeptides linked by bonds not present in natural peptides. The reactionscan then be quantified using standard quantification procedures, todetermine the efficiency of production of the phage library.

The present method can be used to generate enriched phage libraries thatcontain, for example, derivatives that can be synthesized in a sequenceof chemical steps from natural peptides. Each derivative, thus, can beeasily amplified and characterized. The present method can be optimizedfor specific reaction conditions depending on the functional group to beassayed.

Phage display, while a robust procedure, is often performed “blindly”with minimal quantitative monitoring during the process. The presentmethod employs the quantification of derivatized phage-displayedpeptides to produce optimized reaction conditions to improve the yieldof the desired derivatized peptide. Ideally, the method can be used tomonitor the success of the phage display earlier on in the reactionprocess. This reduces the need for bulk biochemical methods whichinherently less sensitive, time consuming, and performed often muchlater in the process.

The present method does not require co-display of any enzymes; theproduction of synthetic libraries can be done from any phage or viraldisplay. The method can generate a phage that displays chemicallymodified peptides. Further, there is no need for translationalmodification within the phage; derivatization of the peptides fordetection and quantification occurs on displayed peptides.

In another aspect of the present invention there is provided a method ofquantifying an amount of a derivatized peptide displayed on a phage byphage display and comprising an N-terminal serine or threonine residue,the method comprising: providing a phage containing a target peptidethereon; reacting the phage containing the target peptide with a firstreagent to derivatize the target peptide, thereby oxidizing the peptideto form a derivatized peptide comprising an aldehyde group, reacting thealdehyde group on the derivatized peptide with a capture agentcomprising a detection marker, thereby incorporating the detectionmarker within the derivatized peptide; and determining an amount of thedetection marker, thereby quantifying the amount of the derivatizedpeptide displayed on the phage.

Another aspect of the present invention is a method for quantificationof diverse reactions of aldehydes displayed on peptide or libraries ofpeptide by monitoring the disappearance of reactive aldehyde group.Reactions described herein are oxime and hydrazine formations, Wittigreaction, cycloadditions with aromatic bis-amines, Dimrothrearrangements. Examples not shown but feasible based on current stateof the art include any other reactions of aldehydes or aldehyde-derivedimines that take place in aqueous media, such as but not limited to:Morita Baylis Hillman Reaction, Petasis reaction, organocatalyticreactions (asymmetric aldol reactions, Barbas-List Aldol reaction) andmetal-catalyzed reactions (aqueous indium-catalyzed allylations,gold-catalyzed alkylation by terminal alkynes).

In accordance with yet another aspect of the present invention there isprovided a method of quantifying an amount of a macrocyclic peptidedisplayed on a phage by phage display, the method comprising: providinga phage containing a target peptide thereon; reacting the phagecontaining the target peptide with a first reagent to derivatize thetarget peptide to form a derivatized peptide, reacting the derivatizedpeptide with ⁻C≡N⁺-biotin, thereby incorporating the ⁻C≡N⁺-biotin withinthe derivatized peptide to form a macrocyclic peptide; and determiningan amount of the detection marker, thereby quantifying the amount of themacrocyclic peptide displayed on the phage.

In accordance with yet another aspect of the present invention, there isprovided a method of quantifying and selecting a two step reactionsequence, such as cyclization, by incorporating a detection marker instep 1 and elimination of the detection marker in step 2 therebyquantifying and selecting both reaction steps.

In accordance with another aspect of the invention, there is provided akit for quantifying an amount of a derivatized peptide displayed on aphage by phage display, the kit comprising: a first reagent for reactingwith a target peptide displayed on the phage to form a derivatizedpeptide, and a capture agent comprising a detection marker forincorporating within the derivatized peptide. The kit may also provide aphage library that has been modified chemically or genetically todisplay a reactive group and can be modified by anyone not trained inthe art of bioconjugation by one-step mixing with reactive agent. Thekit can provide a capture reagent that can assess the amount of reactivegroup before and after modification, thereby providing a simple andaccurate measure of the yield of the modification. In one kit, thelibrary is already reactive and does not require activation; the captureagent allows the user to asses both quality of library (i.e. amount ofreactive groups) and extent of modification (i.e. amount of reactiongroups left after modification). For example, this can includebioconjugation to a specific reactive group already present on phage(e.g., phage has aldehyde and user simply modifies it without concernabout formation of such aldehyde). Another kit in accordance with thepresent invention can be used to perform and quantify any activation(e.g. oxidize the phage to aldehyde and then react the aldehyde).

BRIEF DESCRIPTION OF THE FIGURES

For a better understanding of the present invention, as well as otheraspects and further features thereof, reference is made to the followingdescription which is to be used in conjunction with the accompanyingdrawings, where:

FIG. 1 shows an example of optimization and quantification of chemicalreactions on phage during oxidation of the N-terminal amino acidsdisplayed on phage.

FIG. 2 shows profiles of the effect of oxidation-quencher on reactionefficiency.

FIG. 3 shows an assessment of the kinetics of the oxidation of theN-terminal Ser on phage.

FIG. 4 shows exemplary yields of oxidation to aldehyde and oximeformation.

FIG. 5 shows a schematic for selecting the subset of a library reactivein oxidation and coupling.

FIG. 6 illustrates the synthesis of genetically-encoded chemicallibraries of glycopeptides (A) or macrocyclic peptides (B).

FIG. 7 illustrates kinetics of panning and amplification of peptidederivatives synthesized via oxidation-oxime ligation.

FIG. 8 illustrates a reaction showing optimization of the synthesis ofpeptide derivatives via reduction of disulfides and alkylation ofcysteines.

FIG. 9 illustrates schematics and results from analysis of kinetics ofreduction and alkylation.

FIG. 10 shows a scheme and results of optimization of the linkercoupling and a cyclization reaction on clonal phage.

FIG. 11 shows a scheme for optimization of a cyclization reactions onlibrary phage and differences in reactivity between clonal population ofphage and library of phage.

FIG. 12 illustrates a scheme for the generation of a light responsivelibrary using cyclization with a light responsive linker.

FIG. 13 illustrates results of panning a library cyclized with unnaturallight-sensitive linker.

FIG. 14 illustrates the quantification of reactions, described in FIG.1-7, using other biochemical methods.

FIG. 15 illustrates the detection of the marker with linker usingSDS-page gel electrophoresis.

FIG. 16 illustrates an optimization of the capture of derivatizedpeptides by biotin/streptavidin interactions.

FIG. 17 illustrates an optimization of various aldehyde reaction onphage

FIG. 18 illustrates an example of a step-wise cyclization reaction

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

As used in the specification and claims, the singular forms “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise.

The term “comprising” as used herein will be understood to mean that thelist following is non-exhaustive and may or may not include any otheradditional suitable items, for example one or more further feature(s),component(s) and/or ingredient(s) as appropriate.

The term “assay” is used herein to refer to a test to qualitatively orquantitatively detect the presence of a substance in a sample.

The term “biological sample” is used herein to refer to both animal andhuman body fluids, excreta and tissues obtained from a living or deadorganism. The term “body fluid”, as used herein includes a naturallyoccurring and/or secreted and/or excreted and/or discharged fluid and/orwash fluid from the surface or inside the bodies of a human or an animaland includes, but is not limited to: saliva, sputum, serum, plasma,blood, pharyngeal, nasal/nasal pharyngeal and sinus secretions, urine,mucous, feces, chyme, vomit, gastric juices, pancreatic juices,semen/sperm, cerebral spinal fluid, products of lactation ormenstruation, egg yolk, amniotic fluid, aqueous humour, vitreous humour,cervical secretions, vaginal fluid/secretions, bone marrow aspirates,pleural fluid, sweat, pus, tears, lymph and bronchial or lung lavage.

The terms “body tissue” or “tissue”, as used herein, refer to anaggregate of cells usually of a particular kind together with theirintercellular substance that form one of the structural materials of aplant or an animal and that in animals include, for example, connectivetissue, epithelium, mucosal membrane, muscle tissue, placental tissue,and tissue from liver, intestines, spleen, kidney, brain, heart, nervetissue, and the like. Samples of body tissue can be obtained from livinghumans or animals by a variety of non-limiting methods, such as fineneedle aspirates, scrapings or biopsy tissue or from the remains of deadhumans or animals. The term “tissue” can be used to refer to naturallyoccurring tissue or synthetic tissue.

Biological samples can also include pre-processed foodstuffs includingplants, samples of meats and processed foods, as well as swab samplesfrom environmental sources such as food processing facilities,hospitals, water, soil, and air. Other biological sample types includeisolates/fractions/concentrates of blood (e.g. platelets, red bloodcells, white blood cells or leukocytes), including umbilical cord bloodor placental blood, bone marrow, aspirates, fine needle organ or lesionaspirates, cervical samples, cultured cells, body swabs, or body smears.

The terms “dormant bacteriophage” and “dormant phage” are usedinterchangeably to refer to bacteriophage that is non-infective, butthat will become infective following activation.

As used herein, the term “reporter gene” refers to a constructcomprising a coding sequence attached to heterologous promoter orenhancer elements and whose expression product can be assayed easily andquantifiably when the construct is introduced into bacteria, for examplethrough infection by bacteriophage carrying the reporter gene.

As used herein, a “target peptide” is a peptide of interest displayed onthe surface of phage that is to be derivatized and assayed. As would beunderstood, the surface of a phage can display a plurality of peptides;a target peptide, thus, is one of the plurality of displayed peptideswhich is targeted for derivatization and assaying.

The term “capture agent” as used herein refers to a compound that reactswith a particular functional group on a phage-displayed peptide. Thecapture agent comprises a reactive moiety that reacts with the desiredfunctional group, and a detection marker which is used to quantify thebinding of the capture agent with the peptide. “Detection marker”, thus,refers to a molecule in association with the moiety to form the captureagent, and that is capable of being assayed when the capture agentreacts with and is attached to the desired functional group. Exemplarycapture agents include those comprising reactive moieties that reactwith terminal aldehyde groups on the phage-displayed peptides, forexample. Exemplary detection markers include biotin, fluorescein andmannose.

As used herein, “derivatization” in the context of a “derivatized phagemolecule” or “derivatized peptide” refers to the modification of atarget peptide residue presented on the surface of a phage, prior toreaction with a capture agent. The modification typically involvesreaction of a particular functional group presented on the targetpeptide, such as the terminal peptide residue, thus forming aderivatized phage molecule, such as a derivatized peptide. Thisderivatized peptide is then detected by and reacted with the captureagent.

Phage Display Quantification Process

The quantification process as described herein generally employs twotypes of phage: peptide-displaying phage “P” and control peptide-freephage “C”. P contains an insert in its genome which encodes and displaysa peptide with its coat proteins. P can also represent a library ofsequences (P_(lib)). The present method is designed such that no changein procedure is necessary when characterizing reaction on one phage P ora library of phage P_(lib).

The control phage C is typically genetically similar to P, but lacksinserts in the phage genome and, thus, a lack of DNA sequence encodingpeptides in the coat protein region. It is desired to be able to performindependent quantification of number of P and C phage particles in thesame solution.

Methods known in the art can be used to differentiate P and C phages.For example:

i) P containing LacZ, while C is LacZ-free. The plaques corresponding toP and C are distinguishable by color on plates containing colorimetricreagent X-gal. Any suitable enzyme other than LacZ can be used, andvisualized using known means, such as with a fluorescence substrate.

ii) P containing genes encoding GFP, RFP or other fluorescent reportersin the genome, while C is reporter-free. The reporter does not have tobe physically linked to any proteins of the phage; it simply can only beexpressed in the host cell. The plaques corresponding to P and C can bedistinguished by color or fluorescence on plates as describedpreviously.

iii) P containing antibiotic resistance gene in the phage genome, whileC is resistance-free. The plaques corresponding to P and C can bedistinguished by plating the mixture of phage on two plates, with andwithout the antibiotic. Alternatively, P and C can carry resistance fortwo types of antibiotics and visualized separately.

iv) P comprises a sequence encoding a short peptide sequence such asFLAG recognized by a specific ligand, such as anti-FLAG antibody, whileC is sequence-free. P and C phage can be distinguished by transferringthe colonies onto nitrocellulose paper and probing with the antibody orother ligand.

v) A mixture of P and C can be lysed to isolate DNA and subject toquantification of specific DNA sequence by real-time PCR or otherquantitative methods. Primers for PCR are designed for insert regionwould recognize DNA of only P but not C.

The characterization of the mixture of P and C is beneficial fordetermining the success of the phage display process. Alternatively, Pand C can be modified chemically and quantified in two separatesolutions. For simplicity, in all subsequent discussions, examples ofthe present method as described herein are based on the presence ofreporter Xgal in P but not in C. This method allows for rapidquantification of P and C by counting plaques of specific color (bluefor P and white for C) in a plaque-forming assay.

Analysis and Quantification of Phage Displayed Peptide Derivatives

Characterization of phage libraries can be a challenge, because eachlibrary member is often present in mixture as a single molecule (singleclone). The present method allows for quantification of reaction yieldsin multi-step reactions on complex mixtures of clones (for example, upto 10⁹ different peptides) using a series of capture agents for eachreaction step. The method as described herein employs any knownsubstrate molecule and detection technology that can be conducted onamino-acid sequences.

Quantification and characterization of the chemical reactions in peptidelibraries has been lacking. Existing approaches for quantitativecharacterization by spectroscopic methods (mass-spectrometry, elementalanalysis) and biochemical methods (gel electrophoresis, Western blot,etc) are typically only applicable to large clonal population or phageand viruses (e.g., 10¹⁰-10¹³ particles). They cannot detectmodifications which occur in a population that contains millions ofdifferent phage clones, with each clone present in low amount (e.g.,1-100 copies), The present method provides optimized reaction conditionsfor assaying derivatized peptide libraries.

The present method can also be used for the improvement of yield inchemical reactions conducted on derivatized peptide libraries. In acollection of many peptides, certain peptides undergo a specific type ofthe reaction faster than others. For example, cyclization can occurfaster in peptides of specific conformation, while substitution orelimination can occur in peptides of specific steric and electronicproperties. Although the selection of individual peptide sequences withunique reactivity has been described previously (Barbas, et al., U.S.patent application Ser. No. 08/573,415, filed Dec 15, 1995; Eldridge, G.M., Weiss, G. A. (2011), Hydrazide reactive peptide tags forsite-specific protein labeling. Bioconjugate Chem. 22: 2143-2153), thepresent invention allows for the optimization of multi-step reactions onpeptides to generate a large library (10³-10⁸) of non-peptidicstructures in high yield. Although these libraries are non-peptidic innature, they are amplifiable quantitatively just like parent peptides.Their identities are genetically-encoded and can be deduced from theoriginal peptidic starting materials.

One advantage of the present method is serial optimization of thetwo-step modification of phage libraries. In one example, quantificationof oxidation of N-terminal serine (Ser) and threonine (Thr)electrophilic addition to aldehyde is performed. In another example,quantification of site-selective reduction or disulphide and itsalkylation by light-sensitive linker is shown. However, it would beunderstood that other functional groups, on other amino acids, whetherterminal or internal within the displayed peptide, may be contemplated.The examples shown herein illustrate only certain embodiments of thepresent method.

A library of phage-displayed peptide-derivatives generated with the helpof a quantification approach contains a well-defined number of ligandsof defined structure. In standard phage display, rounds of panning andamplification discover new peptides; in a library of peptide-derivativesrounds of modification, panning and amplification discover newpeptide-derivatives.

To illustrate the above described system and method, an exemplarypanning of peptide-derivatives is shown on model proteins targets, suchas streptavidin (for binding to biotin) and Concanavalin A (for bindingto mannose). However, as would be readily understood that other proteintargets using other capture agents and detection markers can be used.

The phage concentrations [P]₀, [C]₀, [P]_(b4), [C]_(b4), [P]_(af) and[C]_(af) measured in the present method provide information about yieldand specificity of the reaction (initial step 1), as well as itsinterference with viability of phage. Serial variation of the reactionconditions can be then performed to identify the best outcome in thoseparameters.

The present method can be applied to other multi-step reactions. Forexample, multi-step reactions (Rxn1, Rxn2, etc.) can employ differentreaction conditions. Rxn2 converts functional group F1 from Rxn1 toanother functional group F2. To characterize Rxn2, a mixture of phagefrom reaction 1 is exposed to appropriate reagents (Reag2. 1 Reag 2.2,etc). After an appropriate time, the reaction is terminated and theyield concentration of group F2 is determined using capture agent 2(CA2), which contains a group reactive with F2. Exposure to captureagent CA1, reactive to original group F1, can be used to quantify theamount of unreacted groups F1. For example, quantification of thereaction of aldehyde with hydroxylamine can be done by counting phagewith unreacted aldehyde groups, using aldehyde reactive capture agent.Further, quantification of the reaction of thiols (quantification ofunreacted thiols) can be done using thiol-specific capture agent. Also,quantification of the cyclization can be done using chloroacetamidereactive capture agent. Examples of these reactions are describedherein.

Thus, quantification and optimization of a N-step reaction can be doneusing N capture agents to quantify the yield at each step. Changing thecondition at each step optimizes the yield at each step.

The capture agents (CA1, CA2, etc., described above for quantification)can serve another purpose. They can be used to select members of thelibrary that are prone to undergo a particular reaction moreeffectively. This selection can be applied when reactions are conductedon mixture of phage that display library of peptides P^(lib). By runningreaction on P^(lib) and C in specific conditions, the yield can bequantified using capture agent CA1. Unreacted peptides can be separatedfrom those that are reacted. A sub-library containing derivatizedpeptides can be amplified to give a pure subpopulation of phage thatcontain modified ligands only. The present method provides severalexamples of this, including: (1) modification of a random library toselect a sub-library with N-amino acids reactive to oxidation; and (2)modification of a random library of 7 amino acids flanked by twocysteines to select sub-library that can undergo efficient cyclizationwith unnatural linker.

Calculations of Yield and Viability

Yield of the specific reaction is determined as:Y^(s)=100%*[P]_(af)/[P]_(b4)

Yield of the non-specific reaction is determined as:Y^(n)=100%*[C]_(af)/[C]_(b4).

Yield of the reaction that occurs specifically on a peptide, as opposedto any other protein segment of the phage, is determined by comparingY^(s) and Y^(n). Ideally, the Y^(n) is zero, while Y^(s) is close to100%.

The viability of the phage over the course of the reaction is determinedas V=100%*[P]_(b4)/[P]₀. Ideally, the viability of the reaction shouldbe maintained close to 100%, and optimization of the length of reaction,and concentration of reagents, or their presentation can be used tomaximize V. This can result in an increase of the yield with a decreasein viability, as shown herein (see, for example, in FIGS. 1 and 3).

The ability to quantify the yield allows for rapid selection of optimalreaction conditions, which maximizes yield and specificity and minimizesinterference with viability. Parameters that can be varied to optimizethe yield and specificity include: reagent concentration and reactiontime, and the presence of a catalyst. These parameters are exemplifiedherein.

To gain a better understanding of the invention described herein, thefollowing examples are set forth. It should be understood that theseexamples are for illustrative purposes only. Therefore, they should notlimit the scope of this invention in any way.

EXAMPLES Example 1: Two-Step Modification of Phage Libraries

The synthesis of peptide derivatives is a sequence of reactions (forexample, labelled “Rxn1”, “Rxn2”, etc.). In reaction 1, a mixture ofpeptide-displaying phage [P] and control peptide-free phage [C] in knownconcentration [P]₀ and [C]₀ are mixed with appropriate reagents (“Reag1.1”, “Reag 1.2”, etc). After an appropriate time, the reaction isterminated and a capture agent 1 (CA1) is added. The capture agenttypically contains a complimentary reactive group and, for example,biotin. The solution is then mixed with capture support (such as amagnetic bead with a biotin-binding protein streptavidin). Phage thathas undergone the Rxn1 successfully is captured on the support. Thenumbers of phage before capture ([P]_(b4) and [C]_(b4)) and aftercapture ([P]_(af) and [C]_(af)) are recorded.

FIG. 1 shows optimization and quantification of chemical reactions onphage during oxidation of the N-terminal amino acids displayed on phage.Rxn1 comprises the oxidation of an N-terminal serine residue byReag1.1=NaIO₄, which produces an aldehyde moiety. Capture agent CA1 isaldehyde-reactive biotin-hydroxylamine. A phage or library of phage thatpresents peptide with N-terminal Ser or Thr can be oxidized to aldehyde.The amount of the aldehyde and specificity of the oxidation can beassessed using an aldehyde-reactive capture agent (2). A phage is thencaptured using streptavidin-coated magnetic beads. Quantification of thenumber of phage before and after capture can be used to calculate theyield of the reaction. The concentration of reagents and timing ofreactions was used to maximize the yield.

FIG. 1B illustrates a profile of the yield and specificity of thereaction in different reaction conditions. Reactions were performed onmixture of phage clones: (1) phage displaying a peptide with N-terminalSer (“SVEK phage”) and (2) phage containing N-terminal Ala (“WT”). Twophage clones were characterized independently because SVEK containedLacZ reporter and formed blue plaques, while WT formed white plaques.Identical characterization can be performed using a library of clonesthat contain N-terminal Ser.

FIG. 1C illustrates a profile of the viability of the phage inindividual reaction conditions. The method can be used to rapidlyprofile multiple conditions to select those that maximize the yield ofspecific reaction on SVEK phage, minimizes non-specific reaction on WTphage, minimizes reaction time, and maximizes the viability of the phage(see boxed values). As demonstrated herein, the ideal concentration ofNaIO₄ in Rxn1 is 20-500 μM, more particularly 20 μM or 500 μM.

The concentration of biotin-ARP was also optimized to avoid non-specificbinding. Ideally, the concentration of biotin-ARP is about 0.2-5 mM,typically 0.2 mM, 1 mM or 5 mM. The ideal reaction time (Rxn 2) wasfound to be 1-4 hours, typically 1, 2, 3 or 4 hours incubation time. Theaddition of an analine catalyst was found to favourably promote thereactions.

FIG. 2 shows a profile of the effect of an oxidation-quencher onreaction efficiency. The oxidation of N-terminal Ser on phage can beterminated by a variety of reducing agents. The use of some reducingagents (thio vs. sulfide) can dramatically improve the yield.Thiol-based reducing agents provide similar yields, but glutathione hasthe least negative impact on phage viability.

In FIG. 3, the oxidation of N-terminal Ser presented on phage was “finetuned” to produce an optimal yield of captured phage. All reactions wereconducted with 10¹¹ pfu/mL of phage. After each oxidation, the reactionwas terminated with 4-methoxybenzenethiol; the phage was exposed to 1 mMbiotin-hydroxylamine for 1 h, diluted 1,000,000-fold and captured onStrep-MB. The resultant kinetic curves demonstrate that yield of thereaction in these conditions cannot be increased beyond 90% regardlessof oxidant concentration or reaction time (top two panels). At extendedreaction time or high oxidant concentrations, a decrease in viabilitytake place (lower panels). Side reactions, including modification of WTphage, are minimal in these reaction regimes.

FIG. 4 shows a profile of the yield of a two-step reaction: oxidation toaldehyde and oxime formation, by characterizing unreacted aldehydes. Inpanel A, phage was exposed to optimal oxidation conditions (0.06 mMNaIO₄ for 5 min) and quenched with glutathione. The phage was thenexposed to mannose-hydroxylamine. The amount of unreacted aldehydes werequantified by exposure to capture agent biotin-hydroxylamine (panel B).As illustrated in the graph therein, approximately 90% of the phage isreactive towards biotin-hydroxylamine, but only 10% of phage aremodifiable by biotin-hydroxylamine after 1 h exposure to 1 mMmannose-hydroxylamine. The yield of reaction with mannose-hydroxylamineis 90−10=80%. Panel D shows that 20% of the phage in a random library isreactive towards biotin-hydroxylamine after oxidation, confirming that20% of the library contain N-terminal Ser or Thr residues. Panel Eillustrates that the chemical bond on phage is stable for multiple days.

FIG. 5 illustrates a method of selecting a subset of a library reactivein oxidation and coupling to determine the yield of displayed peptidescomprising terminal Ser/Thr residues (“S/T-terminated sub-library) vs.library members not containing terminal Ser/Thr residues (“non-S/T-term.Library members”). In this example, a random library of phage (panel A)was exposed to optimal oxidation conditions (0.06 mM NaIO₄ for 5 min)and quenched with glutathione and oxime-formation conditions (1 mMbiotin-TEV-hydroxylamine). Since only 20% of the random library containsN-terminal Ser, the yield of the reaction on random naive library is˜20%. After release of the captured phage, and re-amplification (panelC), the yield of identical reaction in identical conditions is typicallyhigher, approaching nearly 100% yield after a few rounds of thisselection. This process, thus, selects a subset of library that reactsoptimally in these reaction conditions.

FIG. 7 shows that a library of peptide derivatives synthesized viaoxidation-oxime ligation can be used in panning and amplification.Example of panning of peptide-Mannose conjugates against a modelmannose-binding target, Concanavalin A (panels A and B), and panning ofthe peptide library which has not been modified (panels C and D) areshown. To assess the efficiency of panning, the amount of phage washedat each step was measured (A and C). From these values and the amount ofphage eluted from beads, back-calculation of the amount of phage on-beadat each washing step (B and D) determined the quantity of phage. Thesevalues follow standard first-rate off-rate kinetics. Each library hasbeen deliberately contaminated with insert-free wild type phage, whichcan be quantified in the mixture, because it forms plaques of whitecolor. By contrast, the library forms plaques of blue color. Insert-freephage serves as internal control for non-specific binding. To assess theefficiency of panning, the amount of “blue” and “white” phage washed ateach step was measured. An increased number of phage at the elution step(EL) as compared to the last washing step (wash 10) indicated that thephage was recovered due to non-specific interaction. For more detailsabout blue-white screen in phage-display panning see: R Derda, S Musah,B P Orner, J R Klim, L Li, L L Kiessling “High-throughput Discovery ofSynthetic Surfaces that Support Proliferation of Pluripotent Cells” JAm. Chem. Soc. 2010, 132, 1289.

Example 2: Optimization of Cysteine (Cys)-Containing Residues

FIG. 8 illustrates the optimization of synthesis of peptide derivativesvia reduction and alkylation of phage displayed peptides. (A) Reactionswere performed with a mixture of L-phage that displays disulfideCPARSPLEC and forms blue plaques with WT-phage that displays no peptideand forms white plaques. (B) After exposure of WT+L mixture to iTCEP andBIA, white and blue plaques after reaction (^(WT)N and ^(L)N) and afterthe biotinylated phage is captured by streptavidin (^(WT)N_(s) and^(L)N_(s)) was counted. Capture of L phage (^(L)Cap) was used as measureof biotinylation. Capture of WT phage (^(WT)Cap) typically indicatesnon-specific reduction or alkylation; however, significant capture wasnot observed (see D-G). (C) Biotinylation of five pIII-displayeddisulfides yields phage with 0-10 biotins (B₀-B₁₀) is shown. Reactionyield is calculated from captured fraction, (D) After exposure of WT+Lmixture to iTCEP and BIA, capture of L-phage increases with the time ofreduction and alkylation. (E) Captures were converted to yields usingequation (C) and the data were fit to pseudo-first order kinetics. Thiskinetics was appropriate because [iTCEP] and [BIA]>>[phage] and theyields of reduction (F) and alkylation (G) of phage at a specific timewere independent of the concentration of phage.

FIG. 9 shows one embodiment of tuning the kinetics of reduction andalkylation of the disulfide residues on Cys by TCEP and their subsequentalkylation by BIA. A) Alkylation of phage does not occur in the absenceof reducing agent. Panel B illustrates quantification of yield ofalkylation of phage clone L by BIA after reduction by TCEP. Panel Cindicates reduction by TCEP is complete in <30 minutes. The totalreaction time is X min. Panel D indicates optimization of alkylationtime after 2 h reduction of phage. The total reaction time is X+Y min.Panel E illustrates optimization of one-pot reduction/alkylation ofphage by varying (F) concentration of TCEP and (G) reduction time. Asshown in Panel H, efficiency of one-pot reaction drops to 60% when phageconcentration is <10⁹ PFU/mL due to low TCEP reduction efficiency atthese concentrations.

FIG. 10 illustrates one example of optimization of the linker couplingand cyclization on clonal phage and library phage. In panel A, reductionand alkylation of the disulfide peptide displayed on the phage andpotential resulting by-products is shown. Exposure to (B) BIA is used toquantify the reduced disulfides or (C) unreacted thiols. Panel Dillustrates that exposure to biotin-thiol (BSH) quantified non-cyclizedpeptides. In Panel E, the fraction of biotinylated phage decreases afterincubation of phage with BSBCA. No biotinylation after exposure to BSHindicated that BSBCA coupling yielded cyclic peptide as major product.In Panel F, disappearance of biotinylated phage was fit withpseudo-first order kinetics to calculate the rate constant (k) ofalkylation by BSBCA. In Panel G, two rate constants measured for twophage-displayed peptides (F1, F2) were similar to those measured usingHPLC and a synthetic peptide of the same amino acid sequence (P1, P2).As shown in Panel H, mass spectrometry confirmed that a cyclic productwas major product of reaction between BSBCA and peptideACPARSPLECGGGSAETVESC(Cam)LAKS (underlined sequences is a syntheticfragment of pIII).

Example 3: Cyclization Reaction

Exemplified herein is the generation of large sub-libraries, from randompeptide libraries, that undergo quantitative N-terminal oxidation, andan efficient three-step cyclization via nucleophilic substitution,cyclization and rearrangement through the Ugi reaction.

A selection for sub-library of sequences that can undergo complex,multi-component reactions involving multiple natural side chains. As anexample, the Ugi multi-component reaction was performed. This reactionrequires amine, carboxylic acid, aldehyde and isocyanide (see FIG. 6).Components in the phage library can be oxidized at the N-terminus (Serand Thr-terminated peptides). Only some sequences can undergo efficientcyclization. Using an isocyanide with a capture agent (such as biotin),sequences that undergo efficient cyclization can be separated from thosethat do not. The sub-library can be used to generate a pure library ofphage-displayed macrocycles.

FIG. 6 illustrates one example of selecting for genetically-encodedchemical libraries of glycopeptides (A) or macrocyclic peptides (B).These peptides are derived from aldehyde-containing peptides (D, andintermediate C). The aldehyde displaying libraries can be quantitativelyproduced from any native phage-displayed peptide library; the yield ofthis reaction can be maximized as described in FIG. 5. A key aldehydeintermediate (C) leads to different libraries in a one-step synthesis.The Ugi reaction is shown: addition of ⁻C≡N⁺-biotin initiates a cascadeof cyclization and rearrangement to afford the complex macrocyclicpeptides. Only product that undergoes complete transformationincorporates biotin within it. A sub-library, which undergoes Ugimacrocyclization, can thus be isolated and re-amplified to yield a pure,genetically encoded library of macrocycles.

FIG. 11 illustrates one exemplary scheme for the optimization of libraryon phage. Conditions that are optimal for phage that display individualsequences do not yield the same yields on libraries containing multiplereactive peptides. Panel A illustrates one scheme of the reduction andalkylation reactions. In Panel B, the observed yield of the alkylationin the library is ˜35%—same conditions for clonal population of phageyield >80% alkylated phage (FIG. 10). In Panel C, the expected couplingin oxime modification, based on optimized conditions for clonalpopulation of phage (FIGS. 2 and 3) is ˜80%. The observed yield in thelibrary is 50%. Thus, it has been surprisingly found that conditionsoptimized for clones that display one type of peptide provide much loweryield in large libraries of clones presenting diverse peptides.Differences depend on the specific reaction and vary from subtledifferences 80% (clone) in 60% in library to significant variations >99%(clone) in 50% (library).

FIG. 12 shows an example of the selection of chemically modifiedgenetically-encoded library and general strategy for the synthesis ofgenetically-encoded light-responsive phage libraries. The yield of eachstep (reduction, alkylation, cyclization) can be improved using stepsdescribed herein (FIGS. 7-9) to yield a pure library, in which everymember contains light responsive linker.

FIG. 13 shows the results of panning of a library cyclized with anunnatural light-sensitive linker. In Panel A, a scheme of panningprocedure for modified library against streptavidin is shown. In PanelB, stacked bars show composition of the library before each round of thepanning. Panel C shows enrichment of binding ligands after each round ofpanning represented as the ratio of the number of eluted bound phage tothe number of phage input at each round. Panel D shows a Venn diagramrepresentation of phage population after panning. Area is proportionalto the number of binding/non-binding phage clones; data based on ELISAscreening of 80 clones. As shown in Panel E, iterative irradiation ofL36 and L42 did not cause a significant change in photo-switchingability of the ligands. The * shows that no complex was observed. TheUgi reaction is just one example of a modified sub-library that can bemade. Other reactions may be used as applicable involving aldehyde, suchas Passerini reaction, Petasis reaction, Wittig reaction, or Mukaiyamaaldol reaction.

Example 4: Comparison of Present Method with Other Biochemical Methods

FIG. 14 illustrates the quantification of reactions, described in FIG.1-7, using other biochemical methods. The coupling of fluorescein wasused to visualize which protein of phage the reaction occurs on. Thebands of the SDS page gel, which correspond to minor protein pIII becomeprogressively more fluorescent with increasing concentration offluorophore (using semi-quantitative analysis). Because pIII protein ispresent at much lower abundance compared to other proteins on phage(e.g. pVIII is present at >1000-fold higher concentration), overallinterpretation of gel is difficult. Large amounts of non-specificfluorescence is observed at the bottom of the gel; this may beassociated with major coat protein pVIII, or could correspond to freefluorescein retained on the gel.

FIG. 15 illustrates the detection of the marker with linker usingSDS-page gel electrophoresis. Phage was reacted with fluoresceiniodoacetamide (FIA) before and after the reaction with the linker.Fluorescence after the reaction with linker indicates remainingunreacted groups (ie. an indication of efficiency of the linkercoupling). The gel shows that coupling at lower concentrations of BSBCAlinker does not work even at higher reaction temperatures. At highconcentration of linker (1 mM), the reaction proceed equally well at 55°C. and room temperature.

Thus, it can be seen that the present method offers improved detectionand quantification of derivatized peptides over conventional bulkmethods.

Example 5: Optimization of the Capture of Derivatized Peptides byBiotin/Streptavidin Interactions

FIG. 16 illustrates an optimization of the capture of derivatizedpeptides by biotin/streptavidin interactions. Conditions were selectedunder which biotinylated phage was captured by magnetic bead(MB)-streptavidin quantitatively but capture of non-biotinylated phagewas zero. Additionally, capture of biotinylated phage isbiotin-specific; it does not occur if the MB-streptavidin is pre-blockedwith soluble biotin.

Example 6: Optimization of Various Aldehyde Reactions on Phage

FIG. 17 illustrates the optimization of various aldehyde reactions onphage. In this figure, from top-to-bottom: oxime ligation of complexglycan, aminobenzylamine ligation/Dimroth rearrangement Wittig reaction.In each case, the phage of library was oxidized to form aldehyde. Theamount of aldehyde before and after reaction was characterized usingbiotin-hydroxylamine as described in FIG. 4. “100% conversion” meansthat no reactive aldehyde was detected after the incubation with theappropriate reagent. Conversion of the N-terminal aldehyde to thedescribed structure was confirmed in an independent reaction onsynthetic peptides in the same conditions (pH, concentration, buffer,temperature) monitored by liquid chromatography mass-spectrometry.

FIG. 18 shows an example of a step-wise cyclization reaction. In thefirst step, phage is modified by the linker that contains a detectionmarker, here biotin. In the second step, the detection marker iseliminated. Steps 1 and 2 can be quantified and selected independently.Unreacted molecules after step 1 can be discarded. Raising the pHpromotes the second reaction (cyclization) and simultaneous elution ofphage from beads. Only molecules reacted in step 2 can be carried to thenext round. Provided example is reaction of phage-displayed aldehydewith molecule that contains a group (“group 1”) that reacts with analdehyde in reaction condition 1 (oxime, reacts at pH=4.7) and a leavinggroup, such as ester that reacts in reaction condition 2 (pH>8). Group 1includes a hydroxylamine (or aminooxy group). Other groups that undergoreaction with aldehyde at pH<8 would be suitable, Examples of suchgroups are stabilized phosphor ylides (Wittig reaction),aminobenzamidoximes and aminobenzylamines.

All publications, patents and patent applications mentioned in thisSpecification are indicative of the level of skill of those skilled inthe art to which this invention pertains and are herein incorporated byreference to the same extent as if each individual publication, patent,or patent applications was specifically and individually indicated to beincorporated by reference.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

1. A method of quantifying=derivatized peptides displayed on a phage byphage display, the method comprising: providing a phage containing atarget peptide displayed thereon; reacting the phage containing thetarget peptide with a first reagent to derivatize the peptide to form aderivatized peptide, reacting the derivatized peptide with a captureagent comprising a detection marker, thereby incorporating the detectionmarker within the derivatized peptide; and determining an amount of thedetection marker, thereby quantifying the amount of the derivatizedpeptide displayed on the phage; wherein the detection marker is capableof detection of less than 100 copies of the derivatized peptide.
 2. Themethod of claim 1, wherein the detection marker is biotin, fluoresceinor mannose.
 3. The method of claim 1, wherein the target peptide is apeptide comprising an N-terminal amino acid.
 4. The method of claim 3,wherein the N-terminal amino acid is serine, threonine or cysteine. 5.The method of claim 1, wherein the derivatized peptide is a glycopeptideor a macrocyclic peptide.
 6. The method of claim 1, wherein the firstreagent is NaIO4, iodoacetamide, or (tricarboxyethyl)phosphine (iTCEP).7. The method of claim 6, wherein the concentration of NaIO4 is 20-500μM.
 8. The method of claim 7, wherein the concentration of NaIO4 is 20or 500 μM.
 9. The method of claim 1, wherein the capture agent isbiotin-iodoacetamide (BIA), biotin-ARP, mannose-hydroxylamine, orchloroacetamide.
 10. The method of claim 9, wherein the concentration ofbiotin-ARP is about 0.2-5 mM.
 11. The method of claim 10, wherein theconcentration of biotin-ARP is about 0.2 mM, 1 mM or 5 mM.
 12. Themethod of claim 9, wherein the reaction time of the capture agent withthe derivatized peptide is 1-4 hours.
 13. The method of claim 4 whereinthe target peptide is oxidized to form a derivatized peptide comprisingan aldehyde group.
 14. The method of claim 1 comprising the furthersteps of: reacting the derivatized peptide with a second reagent in asecond reaction which eliminates the detection marker, and determiningan amount of the detection marker after the second reaction, therebyquantifying the amount of the derivatized peptide after the secondreaction.
 15. A method of quantifying derivatized peptide displayed on aphage by phage display comprising the steps of: providing a phagecontaining a target peptide displayed thereon; in a first reaction,reacting the phage containing the target peptide with a first reagent toderivatize the peptide to form a first derivatized peptide having afirst functional group (F1), in a second reaction, reacting the firstderivatized peptide with a first capture agent (CA1) to react F1 to asecond functional group (F2), wherein the first capture agent is coupledto a first detection marker, reacting the derivatized peptides with asecond capture agent (CA2) coupled to a second detection marker, whereinCA2 is reactive with F1 and the second detection marker is differentfrom the first detection marker, determining an amount of the firstdetection marker to quantify the amount of the derivatized peptidebearing F2, and determining an amount of the second detection marker toquantify the amount of derivatized peptide bearing F1.
 16. The method ofclaim 18 wherein the first reagent oxidizes an N-terminal amino acid toproduce an aldehyde moiety, and the second reagent converts the aldehydemoiety to an oxime.
 17. The method of claim 18 wherein the first reagentreduces disulfide bonds and CA1 alkylates the peptides.
 18. A method ofselecting for a first genetically encoded library of first derivatizedpeptides displayed on a phage (A) or a second genetically encodedlibrary of second derivatized peptides displayed on a phage (B),comprising the steps of: providing phages containing a target peptidedisplayed thereon; producing a library of intermediate derivatizedpeptides from the target peptides, reacting the intermediate derivatizedpeptides with a capture agent coupled with a detection marker in areaction which, if complete, results in the first derivatized peptidesdisplaying the detection marker, and if incomplete, results in thesecond derivatized peptides not displaying the detection marker,isolating the first derivatized peptides using the detection marker. 19.The method of claim 21 wherein the target peptides are reacted toproduce intermediate derivatized peptides having terminal aldehydemoieties.
 20. The method of claim 22 wherein the capture agent coupledwith a detection marker comprises ⁻C≡N⁺-biotin, which reacts with thealdehyde moiety to produce a macrocyclic peptide, if complete.